1H, 13C, and 15N resonance assignment of the central domain of hRSV transcription antitermination factor M2-1.
ABSTRACT M2-1 is an essential co-factor of the respiratory syncytial virus, an important respiratory pathogen in infants and calves. It acts as a transcription antitermination factor which enhances the processivity of the polymerase. Within the polymerase complex, M2-1 interacts with a second co-factor, the phosphoprotein P. It has been shown previously that P and RNA bind to M2-1 in a competitive manner in vitro and that these properties are related to a central domain located between residues Glu59 and Lys177. Here we report the almost complete (1)H, (13)C and (15)N assignment of a fragment of M2-1 corresponding to this region, for further structure determination and interaction studies.
- SourceAvailable from: Mohammed Moudjou[show abstract] [hide abstract]
ABSTRACT: The respiratory syncytial virus (RSV) M2-1 protein is an essential cofactor of the viral RNA polymerase complex and functions as a transcriptional processivity and antitermination factor. M2-1, which exists in a phosphorylated or unphosphorylated form in infected cells, is an RNA-binding protein that also interacts with some of the other components of the viral polymerase complex. It contains a CCCH motif, a putative zinc-binding domain that is essential for M2-1 function, at the N terminus. To gain insight into its structural organization, M2-1 was produced as a recombinant protein in Escherichia coli and purified to >95% homogeneity by using a glutathione S-transferase (GST) tag. The GST-M2-1 fusion proteins were copurified with bacterial RNA, which could be eliminated by a high-salt wash. Circular dichroism analysis showed that M2-1 is largely alpha-helical. Chemical cross-linking, dynamic light scattering, sedimentation velocity, and electron microscopy analyses led to the conclusion that M2-1 forms a 5.4S tetramer of 89 kDa and approximately 7.6 nm in diameter at micromolar concentrations. By using a series of deletion mutants, the oligomerization domain of M2-1 was mapped to a putative alpha-helix consisting of amino acid residues 32 to 63. When tested in an RSV minigenome replicon system using a luciferase gene as a reporter, an M2-1 deletion mutant lacking this region showed a significant reduction in RNA transcription compared to wild-type M2-1, indicating that M2-1 oligomerization is essential for the activity of the protein. We also show that the region encompassing amino acid residues 59 to 178 binds to P and RNA in a competitive manner that is independent of the phosphorylation status of M2-1.Journal of Virology 05/2009; 83(13):6363-74. · 5.08 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: NMR chemical shifts in proteins depend strongly on local structure. The program TALOS establishes an empirical relation between 13C, 15N and 1H chemical shifts and backbone torsion angles phi and psi (Cornilescu et al. J Biomol NMR 13 289-302, 1999). Extension of the original 20-protein database to 200 proteins increased the fraction of residues for which backbone angles could be predicted from 65 to 74%, while reducing the error rate from 3 to 2.5%. Addition of a two-layer neural network filter to the database fragment selection process forms the basis for a new program, TALOS+, which further enhances the prediction rate to 88.5%, without increasing the error rate. Excluding the 2.5% of residues for which TALOS+ makes predictions that strongly differ from those observed in the crystalline state, the accuracy of predicted phi and psi angles, equals +/-13 degrees . Large discrepancies between predictions and crystal structures are primarily limited to loop regions, and for the few cases where multiple X-ray structures are available such residues are often found in different states in the different structures. The TALOS+ output includes predictions for individual residues with missing chemical shifts, and the neural network component of the program also predicts secondary structure with good accuracy.Journal of Biomolecular NMR 07/2009; 44(4):213-23. · 2.85 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: Human respiratory syncytial virus (RSV) is the leading cause of paediatric respiratory disease and is the focus of antiviral- and vaccine-development programmes. These goals have been aided by an understanding of the virus genome architecture and the mechanisms by which it is expressed and replicated. RSV is a member of the order Mononegavirales and, as such, has a genome consisting of a single strand of negative-sense RNA. At first glance, transcription and genome replication appear straightforward, requiring self-contained promoter regions at the 3' ends of the genome and antigenome RNAs, short cis-acting elements flanking each of the genes and one polymerase. However, from these minimal elements, the virus is able to generate an array of capped, methylated and polyadenylated mRNAs and encapsidated antigenome and genome RNAs, all in the appropriate ratios to facilitate virus replication. The apparent simplicity of genome expression and replication is a consequence of considerable complexity in the polymerase structure and its cognate cis-acting sequences; here, our understanding of mechanisms by which the RSV polymerase proteins interact with signals in the RNA template to produce different RNA products is reviewed.Journal of General Virology 08/2006; 87(Pt 7):1805-21. · 3.13 Impact Factor